CRISPR-ON-Mediated KLF4 overexpression inhibits the proliferation, migration and invasion of urothelial bladder cancer in vitro and in vivo

Abstract

Kruppel like factor 4 (KLF4), a transcription factor associated with carcinogenesis and tumor progression, plays an important role in various malignancies. In the present study, we utilized the CRISPR-ON system to upregulate KLF4 expression level and subsequently investigated the effect and mechanism of KLF4 in the carcinogenesis and progression of urothelial bladder cancer (UBC). Immunohistochemistry (IHC) and quantitative RT-PCR (qRT-PCR) were used to evaluate the expression of KLF4. The CpG methylation status of the promoter region was analyzed using bisulfite-sequencing PCR (BSP). CRISPR-ON system comprised sgRNA and dCas9 protein combined with a transcriptional activation domain. The cell proliferation and cell cycle were assessed by CCK-8 assay, flow cytometry and colony formation assay. The cell motility ability was evaluated using trans-well assay. In vivo tumorigenesis assay and lung metastasis model were also performed. The KLF4 expression was significantly downregulated in UBC tissues. The high CpG methylation status in the promoter of KLF4 was confirmed using BSP. KLF4 overexpression was successfully achieved via CRISPR-ON system, which inhibited the proliferation and induced G1-phase arrest in T24 cells through the regulation of AKT/p21 signal. Furthermore, enforced expression of KLF4 also abrogated the migration and invasion of T24 cells by suppressing EMT progression. Finally, in vivo models indicated that the upregulation of KLF4 could inhibit tumorigenesis and lung metastasis in nude mice. In conclusion, KLF4 overexpression mediated by CRISPR-ON inhibits tumorigenesis and EMT progression in UBC cells, representing a potential therapeutic target, and CRISPR-ON system could be a therapeutic strategy for UBC in the future.

METTL3/YTHDF2 m6A axis promotes tumorigenesis by degrading SETD7 and KLF4 mRNAs in bladder cancer

“…The important transcription factor KLF4, acted as a suppressor in metastasis and proliferation of cancer cells in colorectal cancer, while driven expression of KLF4 in tumour‐associated macrophages promoted immunosuppression to facilitate tumour growth in glioblastoma . However, by means of this study and our previous research, we demonstrated that SETD7 and KLF4 were two evident tumour suppressor genes in BCa. Consequently, the degradation of tumour suppressor genes by the m 6 A regulatory axis probably plays a vital role in the tumorigenesis of BCa.…”

“…The decreased expression was also observed in distinct cancerous cell lines (Figure C). Our previous studies have demonstrated the explicit anti‐cancer role of KLF4 in BCa, which has also been acknowledged in other urologic cancers, such as prostate cancer and renal cancer . Therefore, we focused on exploring the mechanism of SETD7.…”

“…Among the 142 up‐regulated genes identified in METTL3‐depleted T24 cells (log 2 FC > 1 and P ‐value < .05), 46 genes were predicted to be negatively correlated with METTL3 in the TCGA database (Figure D). Integrating the results of the database LinkedOmics (http://www.linkedomics.org), the specific function induced by METTL3 and previous studies of our research group, SET domain containing 7 (SETD7, a histone lysine methyltransferase) and Kruppel‐like factor 4 (KLF4, an accepted transcription factor) were selected as the principal potent downstream targets (Figure E).…”

Long non‐coding RNA LINC00673 silencing inhibits proliferation and drug resistance of prostate cancer cells via decreasing KLF4 promoter methylation

“…In line with our findings, Xu et al further demonstrated that KLF4 was poorly expressed, while the CpG islands were highly methylated in the KLF4 gene promoter in urothelial bladder cancer tissues 29. Furthermore, lncRNA PVT1, which was elevated in prostate cancer, was reported to serve as an oncogene in prostate cancer by promoting the methylation of mi-croRNA-164a 30.…”

“…Furthermore, depleted expressions of KLF4 have been detected in other cancers, such as breast cancer,25 while another study reported that overexpression of KLF4 could repress the growth and invasion of cancer cell lines, including cell lines of prostate cancer 26. In line with our findings, Xu et al further demonstrated that KLF4 was poorly expressed, while the CpG islands were highly methylated in the KLF4 gene promoter in urothelial bladder cancer tissues 29. This specific process of abnormal DNA methylation is associated with gene expression,27 and another study also noted that the expression of KLF4 could be depleted by promoter methylation in classical Hodgkin lymphoma cells 28.…”